EFFECT OF PERIODATE-OXIDIZED ATP AND OTHER NUCLEOTIDES ON FIREFLY LUCIFERASE

Addition of periodate-oxidized ATP (oATP) to firefly luciferase-contai ning reaction mixtures enhanced light production when the reaction mix ture contained > similar to 8 mu M ATP. The time course of light produ ction was changed from a flash pattern to a constant light output duri ng incubations of < similar to 10 min. During longer incubation, firef ly luciferase was inactivated in a concentration-dependent fashion by oATP. Firefly luciferase has two different time courses of Light produ ction that depend on ATP concentration (DeLuca and McElroy, Biochem. B iophys. Res. Commun,, 123, 764, 1984). The enhancement of light produc tion occurred only when higher ATP concentrations (> 8 mu M) were used . There is little effect of oATP on firefly luciferase activity at low ATP concentrations (< 2 mu M) which gave steady production of light. ATP did not antagonize the inactivation of firefly luciferase by oATP. When the oATP was chemically reduced with sodium borohydride (giving orATP), there was no inactivation of firefly luciferase on incubation. When orATP was used in a short incubation the enhancement of light pr oduction and time course change were the same as those observed with o ATP. The corresponding AMP and adenosine compounds (o and or) were sli ghtly inhibitory to firefly luciferase activity. ADP was without effec t but both oADP and orADP enhanced light production. Of these periodat e-oxidized ADP, AMP, and adenosine derivatives only oADP inactivated f irefly luciferase. The activating effect can be explained by a change in the conformation of the enzyme-product complex so that the product is released faster. In addition there is an inactivation of the enzyme by certain periodate-oxidized nucleotides during longer incubations, (C) 1994 Academic Press, Inc.